Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
This invention pertains to the shuffling of nucleic acids to achieve or enhance industrial production of chemicals by monooxygenase genes.
Organic acids, alcohols, aldehydes and epoxides are important classes of industrial chemicals. Typically, these products are generated by successive oxidation of inexpensive, high volume saturated and unsaturated hydrocarbons (ethane, propane, butane, etc. and ethene, propene, butene, etc.) and simple aromatics such as benzene, ethyl benzene, naphthalene, styrene and toluene.
Monooxygenases (MOs) such as the P450 oxygenases, heme-dependent peroxidases, iron-sulfur MOs and quinone-dependent MOs typically catalyze limited oxidation of these basic chemical building blocks. While potentially interesting from an industrial standpoint, these enzymes typically exhibit neither the physical robustness nor sufficient turnover numbers to make them usable as industrial catalysts. In addition, regeneration of a reduced heme is required following each catalytic turnover. Biologically, the necessary heme reduction is mediated in the P450 family of enzymes by NAD(P)H, an expensive and impractical redox partner for most industrial chemistries.
Surprisingly, the present invention provides a method for providing enzymes with higher activity, high physical stability and robustness. Also surprisingly, the present invention provides a means of generating NADPH-independent monooxygenase activity in the presence of peroxide co-substrates (as well as other inexpensive cofactors) thereby solving each of the problems outlined above, as well as providing a variety of other features which will be apparent upon review.
In the present invention, DNA shuffling is used to generate new or improved monooxygenase genes. These monooxygenase genes are used to provide monooxygenase enzymes, especially for industrial processes. These new or improved genes have surprisingly superior properties as compared to naturally occurring monooxygenase genes.
In the methods for obtaining monooxygenase genes, a plurality of parental forms (homologs) of a selected nucleic acid are recombined. The selected nucleic acid is derived either from one or more parental nucleic acid(s) which encodes a monooxygenase enzyme, or a fragment thereof, or from a parental nucleic acid which does not encode monooxygenase, but which is a candidate for DNA shuffling to develop monooxygenase activity. The plurality of forms of the selected nucleic acid differ from each other in at least one (and typically two or more) nucleotides, and, upon recombination, provide a library of recombinant monooxygenase nucleic acids. The library can be an in vitro set of molecules, or present in cells, phage or the like. The library is screened to identify at least one recombinant monooxygenase nucleic acid that exhibits distinct or improved monooxygenase activity compared to the parental nucleic acid or nucleic acids.
Many formats for libraries of nucleic acids are known in the art and each of these formats is generally applicable to the libraries of the present invention. For example, basic texts generally disclosing library formats of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).
In a preferred embodiment, the starting DNA segments are first recombined by any of the formats described herein to generate a diverse library of recombinant DNA segments. Such a library can vary widely in size from having fewer than 10 to more than 105, 107, or 109 members. In general, the starting segments and the recombinant libraries generated include full-length coding sequences and any essential regulatory sequences, such as a promoter and polyadenylation sequence, required for expression. However, if this is not the case, the recombinant DNA segments in the library can be inserted into a common vector providing the missing sequences before performing screening/selection.
If the sequence recombination format employed is an in vivo format, the library of recombinant DNA segments generated already exists in a cell, which is usually the cell type in which expression of the enzyme with altered substrate specificity is desired. If sequence recombination is performed in vitro, the recombinant library is preferably introduced into the desired cell type before screening/selection. The members of the recombinant library can be linked to an episome or virus before introduction or can be introduced directly. In some embodiments of the invention, the library is amplified in a first host, and is then recovered from that host and introduced to a second host more amenable to expression, selection, or screening, or any other desirable parameter.
The manner in which the library is introduced into the cell type depends on the DNA-uptake characteristics of the cell type (e.g., having viral receptors, being capable of conjugation, or being naturally competent). If the cell type is not susceptible to natural and chemical-induced competence, but is susceptible to electroporation, one preferably employs electroporation. If the cell type is not susceptible to electroporation as well, one can employ biolistics. The biolistic PDS-1000 Gene Gun (Biorad, Hercules, Calif.) uses helium pressure to accelerate DNA-coated gold or tungsten microcarriers toward target cells. The process is applicable to a wide range of tissues, including plants, bacteria, fungi, algae, intact animal tissues, tissue culture cells, and animal embryos. One can employ electronic pulse delivery, which is essentially a mild electroporation format for live tissues in animals and patients. Zhao, Advanced Drug Delivery Reviews 17:257-262 (1995). Novel methods for making cells competent are described in co-pending application U.S. patent application Ser. No. 08/621,430, filed Mar. 25, 1996. After introduction of the library of recombinant DNA genes, the cells are optionally propagated to allow expression of genes to occur.
In selecting for monooxygenase activity, a candidate shuffled DNA can be tested for encoded monooxygenase activity in essentially any synthetic process. Common processes that can be screened include screening for alkane oxidation (e.g., hydroxylation, formation of ketones, aldehydes, etc.), screening for alkene epoxidation, aromatic hydroxylation, N-dealkylation (e.g., of alkylamines), S-dealkylation (e.g., of reduced thio-organics), O-dealkylation (e.g., of alkyl ethers), oxidation of aryloxy phenols, conversion of aldehydes to acids, alcohols to aldehydes or ketones, dehydrogenation, decarbonylation, oxidative dehalogenation of haloaromatics and halohydrocarbons, Baeyer-Villiger monoxygenation, modification of cyclosporins, hydroxylation of mevastatin, hydroxylation of erythromycin, N-hydroxylation, sulfoxide formation, hydroxylation of fatty acids, hydroxylation of terpenes or oxygenation of sulfonylureas. Other oxidative transformations will be apparent to those of skill in the art.
Similarly, instead of, or in addition to, testing for an increase in monooxygenase specific activity, it is also desirable to screen for shuffled nucleic acids which produce higher levels of monooxygenase nucleic acid or enhanced or reduced recombinant monooxygenase polypeptide expression or stability encoded by the recombinant monooxygenase nucleic acid.
A variety of screening methods can be used to screen a library, depending on the monooxygenase activity for which the library is selected. By way of example, the library to be screened can be present in a population of cells. The library is selected by growing the cells in or on a medium comprising the chemical or compound to be oxidized or reduced and selecting for a detected physical difference between the oxidized or reduced form of the chemical or compound and the non-oxidized or reduced form of the chemical or compound, either in the cell, or the extracellular medium.
Iterative selection for monooxygenase nucleic acids is also a feature of the invention. In these methods, a selected nucleic acid identified as encoding monooxygenase activity can be shuffled, either with the parental nucleic acids, or with other nucleic acids (e.g., mutated forms of the selected nucleic acid) to produce a second shuffled library. The second shuffled library is then selected for one or more form of monooxygenase activity, which can be the same or different than the monooxygenase activity previously selected. This process can be iteratively repeated as many times as desired, until a nucleic acid with optimized properties is obtained. If desired, any monooxygenase nucleic acid identified by any of the methods herein can be cloned and, optionally, expressed.
The invention also provides methods of increasing monooxygenase activity by whole genome shuffling. In these methods, a plurality of genomic nucleic acids are shuffled in a cell (in whole cell shuffling, entire genomes are shuffled, rather than specific sequences). The resulting shuffled nucleic acids are selected for one or more monooxygenase traits. The genomic nucleic acids can be from a species or strain different from the cell in which monooxygenase activity is desired. Similarly, the shuffling reaction can be performed in cells using genomic DNA from the same or different species, or strains. Strains or enzymes exhibiting enhanced MO activity can be identified.
The distinct or improved monooxygenase activity encoded by a nucleic acid identified after shuffling can encode one or more of a variety of properties, including: an increased ability to chemically modify the monooxygenase target, an increase in the range of monooxygenase substrates which the distinct or improved nucleic acid operates on, an increase in the chemoselectivity of a polypeptide encoded by the nucleic acid, an increase in the regioselectivity of a polypeptide encoded by the nucleic acid, an increase in the stereoselectivity of a polypeptide encoded by the nucleic acid, an increased expression level of a polypeptide encoded by the nucleic acid, a decrease in susceptibility of a polypeptide encoded by the nucleic acid to protease cleavage, a decrease in susceptibility of a polypeptide encoded by the nucleic acid to high or low pH levels, a decrease in susceptibility of the protein encoded by the nucleic acid to high or low temperatures, a decrease in peroxide-mediated enzyme inactivation, a decrease in toxicity to a host cell of a polypeptide encoded by the selected nucleic acid, the ability to use low-cost reducing partners (rather than NAD(P)H), and a reduction in the sensitivity of the polypeptide and/or an organism expressing the polypeptide to inactivation by organic solvents and the feedstocks for and products of the enzymatic oxidations, and
The selected nucleic acids to be shuffled can be from any of a variety of sources, including synthetic or cloned DNAs. Exemplary targets for recombination include nucleic acids encoding P450 monooxygenases, nucleic acids encoding heme-dependent peroxidases, nucleic acids encoding iron sulfur monooxygenases, nucleic acids encoding quinone-dependent monooxygenases, and the like. Typically, shuffled nucleic acids are cloned into expression vectors to achieve desired expression levels.
In addition to shuffling monooxygenase nucleic acids, it is occasionally desirable to produce shuffled nucleic acids which produce oxidizing/reducing equivalents in forms other than O2, H2O2 and NADPH, such as peroxides. Shuffled monooxygenase and oxidase (H2O2) nucleic acids can be co-expressed in a single system to provide both monooxygenase activity and peroxide in a single system.
One feature of the invention is production of libraries and shuffling mixtures for use in the methods as set forth above. For example, a phage display library comprising shuffled forms of a nucleic acid is provided. Similarly, a shuffling mixture comprising at least three homologous DNAs, each of which is derived from a nucleic acid encoding a polypeptide or polypeptide fragment is provided. These polypeptides can be, for example, P450 monooxygenases, heme-dependent peroxidases, iron sulfur monooxygenases, quinone-dependent monooxygenases, and the like.
Isolated nucleic acids identified by selection of the libraries in the methods above are also a feature of the invention.